JPH0731196B2 - Cell identification method in complex antigen-antibody reaction system and cell immobilization apparatus used in this method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial field of application) The present invention relates to cells having various antigens and cells having an antigen-antibody complex formed by combining antigens with antibodies from a liquid containing various kinds of antibodies. The present invention relates to a cell identification method for identification and an apparatus used for this method.

(Prior Art) Conventionally commonly used immune reaction detection methods are as follows.

Specific methods such as sedimentation method, overlay method, capillary tube method, test tube method, immunodiffusion method, etc., quantitative precipitation method, laser nephelometry, quantitative immunodiffusion method (SRID, RSRID, immunoelectrodiffusion method, cross immunoelectricity method) There is a quantitative method such as electrophoresis.

Hemolysis, lysis method Aggregation method; cell agglutination method, antiglobulin test (Cooms test), passive hemagglutination method (PHA, HA) immunoadhesion method (IA)
Etc.

Passive skin anaferax method (PCA) labeled antibody method; immunofluorescence method (FIA), enzyme immunoassay method (EI)
A) Radioisotope labeling method (RIA) is available.

(Problems to be Solved by the Invention) In each of these methods, various improvements are added depending on the application, and automation and omission are achieved. However, in a general immune reaction, there are many cases where a highly specific monoclonal antibody or complement is not present, or the complement activating function is not effectively used even if complement is present. In such a case, the background rise due to nonspecific reaction and other factors cannot be ignored, and it becomes extremely difficult to identify the target specific reaction.

For example, the cytotoxicity test (LCT) of histocompatibility factor (HLA) based on lysate and lysis method has the following problems. In this test, antiserum derived from a multiparous woman having various and various specificities and antibody titers is used as an antibody, and this antibody is reacted with a sample lymphocyte surface HLA antigen to identify lymphocytes. In this case, a complement that enhances the lymphocyte cell membrane damage function specifically under the influence of the antigen-antibody complex produced as a result of the reaction and a dye that stains the destroyed lymphocytes are used. Then, the degree of specificity of lymphocytes is evaluated from the degree of dye staining that is proportional to the frequency of cell damage. However, when the antigen concentration is low, such as the state where the antigen is dispersed on the surface of 1 lymphocyte, the complement activation ability does not sufficiently act. In addition, complement activation is not observed in the 1gG4 subclass antibody reaction. In such cases, it is extremely difficult to understand the specificity of lymphocytes. Furthermore, although more than 140 HLA antigens have been discovered, the types of predicted antigens are expected to greatly exceed this number, but means for immobilization have not been established.

It is an object of the present invention to identify cells that have an antigen-antibody complex and suppress background by suppressing non-specific reaction without using complement.

[Structure of the Invention] (Means for Solving the Problems) According to the identification method of the present invention, a substance that specifically binds to an immunoglobulin is fixed to a part of the inner wall of youkai, and an antigen and an antibody are bound to this container. A second liquid having a higher specific gravity than the first liquid containing cells having the antigen-antibody complex formed on the surface thereof is contained, and the substance is immersed in the second liquid,
Next, the first liquid is contained on the second liquid, and the container is rotatably moved in a state where the part of the inner wall is farthest from the rotation center, and the cells are treated with the substance. It is bound to and solidified to form a solid phase, which is then washed to assimilate.

The device of the present invention comprises a container, a substance fixed to a part of the inner wall of the container and specifically binding to an immunoglobulin, and a cell having an antigen-antibody complex formed by binding an antigen and an antibody on the surface thereof. The second liquid having a higher specific gravity than that of the first liquid and accommodated in the container so as to immerse the substance, and the container in a state where the part of the inner wall is located farthest from the center of rotation. And a container rotating means for rotating the container.

(Operation) In the identification method of the present invention, a cell in the first liquid has an antigen-antibody complex on its surface. This complex is composed of an antigen existing on the surface of a cell and an immunoglobulin (antibody) bound to this antigen. Cells having such an antigen-antibody complex are suspended in the first liquid. Next, when the container is rotated, the cells having the antigen-antibody complex are subjected to a centrifugal force, enter the second liquid, and approach the substance fixed to the inner wall of the container. The substance then binds to the immunoglobulin FC portion that forms the antigen-antibody complex bound to the cells. Thus, the cells are immobilized on the inner wall of the container.

Here, the first liquid contains immunoglobulin (II) different from the immunoglobulin (immunoglobulin (I)) and cells (II) different from the cells (cell (I)). Shall not be combined with each other. Even in this case, only cells (I) can be immobilized on the inner wall. The reason is as follows. First, since immunoglobulin (II) is much smaller than cells (I), its diffusion rate is slow. Therefore, the cell (I) reaches the substance faster. Therefore, the two can be distinguished from each other if the rotation of the container is stopped before the immunoglobulin (II) reaches the substance. The same applies to the remaining immunoglobulin (I). Meanwhile, cells (II)
When the container rotates, it approaches the substance like the cell (I) by the centrifugal force. However, since the cell (II) does not have an immunoglobulin that binds to the substance, it simply contacts the substance and the inner wall of the container around the substance, and is easily separated from the substance if washed. .

In the apparatus according to the present invention, the first liquid and the second liquid are contained in a container, and the container is rotated by the container rotating means. At this time, the cells having the antigen-antibody complex on their surface are immobilized on the substance fixed to the container. The description up to the solid phase has already been made in the description of the above method, and therefore will be omitted here.

(Example) An example of the identification method of the present invention will be described together with an apparatus used for the method.

FIG. 1 shows the entire apparatus of this embodiment. The drum 1 is supported by the shaft 3 via four support members 2. Both ends of the shaft 3 are supported by a pair of arms 4 and are rotatable. The arm 4 is supported by an arm support base 5. The arm support 5 is fixed to the base 6. The arm support 5 is a housing, and the motor is housed inside. A gear 7 is attached to the rotary shaft of this motor.
A gear 8 is provided on the outer periphery of the drum 1, and the gear 7 meshes with the gear 8. 9 is a controller. The controller 9 controls the rotation speed of the motor. The controller 9 is also attached to the base 6. One or more holders 10 are mounted adjacent to the inner wall of the drum 1. The holder 10 detachably holds a plate 12 having a large number of wells 11 as shown in FIG. As shown in FIG. 3, each well 11 has protein A30 fixed in layers on the inner wall of the bottom surface to be solid-phased.

In this example, predetermined lymphocytes contained in plasma fluid are immobilized. Therefore, as shown in FIG. 3, the liquid 13 having a higher specific gravity than the predetermined lymphocytes is contained in the well 11 of the plate 12. Plasma liquid 14 to be tested is liquid 13
Is held on. The plasma liquid 14 contains serum component 15, blood cell component 16, A antibody 17, B antibody 18, A lymphocyte 19, and B lymphocyte 20. Here, serum component 15 and blood cell component 16
Is a blood component that is not directly involved in the immune response. A antibody 17
And A lymphocyte 19 are non-specific in the immune response,
B antibody 18 and B lymphocyte 20 are specific in the immune response. That is, the B lymphocyte 20 causes an antigen-antibody reaction with a part of the B antibody 18 and binds to the B antibody 18. In this example, plasma liquid containing the same lymphocytes is contained in each well 11, and different antibodies are added to each well 11 to assimilate the lymphocytes in which an antigen-antibody reaction has occurred. One plate 12 is provided with 96 wells 11. As shown in FIG. 3, each well 11 of each pool 12 is filled with a liquid 13 having a higher specific gravity than the plasma liquid 14 and the plasma liquid 14. As is apparent from the state shown in FIG. 3, the operator first injects the high specific gravity liquid 13 into the well 11 and then the plasma liquid 14. This is because unless this is done, the antibody in the plasma liquid 14 is captured by the protein A30 immobilized in the well 11 before the drum 1 is rotated. The operator holds one or more plates 12 in the holder 10 shown in FIG. 1 and operates the controller 9 to rotate the drum 1. The controller 9 controls the rotational speed of the drum 1 from the first rotation to the final rotation so that the positional relationship between the plasma liquid 14 and the liquid 13 in the well 11 is maintained.

The following phenomenon occurs in the well 11 while the drum 1 is rotating. First, the A lymphocytes 19 and the lymphocytes 20 pass through the liquid 13 and reach the protein A30. Here, A lymphocyte 19 is only in contact with protein A30, but B lymphocyte 20 is bound with B antibody 18, and this B antibody is
18 FC parts are connected to Protein A30. On the other hand, serum component 15, blood cell component 16, A antibody 17, B antibody 18 (B lymphocyte
The residue that did not bind to 20 has a slower diffusion rate than A lymphocytes 19 and B lymphocytes 20 or has a lower specific gravity, so that A lymphocytes 19 and B lymphocytes 20 have protein A30.
By the time it reaches, the liquid 13 is still in the upper layer.

When the controller 9 rotates the drum 1 for a preset time, it stops it. Operator is Protein A30
The liquid 13 and the plasma liquid 14 are removed from the well 11 to which is adhered, and the plate 12 is allowed to stand for a predetermined time. Well at this time
As shown in FIG. 4, B lymphocytes 20 are captured by protein A30 via B antibody 18 around the bottom of 11 and A
Lymphocytes 19 are in contact with the bottom of well 11. The operator then washed the bottom of well 11 to remove A lymphocytes 19,
Try to leave 20 B lymphocytes. In this way, the B lymphocyte 20 is immobilized.

According to the present example, the predetermined lymphocytes can be solidly immobilized from the plasma liquid, and the apparatus used in this method can surely identify the predetermined lymphocytes.

In this embodiment, the substance adhered to a part of the inner wall of the container is protein A, but it may be another substance, for example protein G, as long as it specifically binds to immunoglobulin.

Further, in the present example, the cells to be assimilated are lymphocytes, which may be erythrocytes having any of the ABO type, MN type, and Rh type antigens, or platelets having the histocompatibility factor HLA antigen. good. However, when immobilizing these cells, it is necessary to remove cells having a greater specific gravity than these cells, such as lymphocytes, from the plasma solution in advance.

In the apparatus of this embodiment, the well 11 has a columnar shape and a semi-spherical bottom, but the shape of the well 11 may be any shape as shown in FIGS. 5 (a) to (i). In FIG. 5, (a) is a truncated cone shape, (b) is an inverted shape, (c)
Is semi-ellipsoidal, (d) is spherical, (e) is conical, (f)
Has a conical shape with an ellipsoidal tip, (g) has a cylindrical shape, (h) has a prismatic shape, and (i) has a pyramidal shape.

Further, although the plate 12 has a quadrangular plate shape in the apparatus of this embodiment, this shape may be a circular plate shape or a polygonal plate shape. Alternatively, these plates may be stacked in layers.

Further, in the apparatus of this embodiment, the drum 1 has a cylindrical shape, but it may have a polygonal cylindrical shape as shown in FIG. 6 (a) or a coaxial shape as shown in FIG. 6 (d). Alternatively, a plurality of drums may be stacked. Further, as shown in FIGS. 6 (b) and 6 (c), a supporting member 40 may be provided so as to project from the shaft 3, and the plate 12 may be held on the tip side thereof.

In the apparatus of this embodiment, the plate 12 was rotated so that a circular orbit was drawn along the vertical plane, but the plate 12 may be rotated as shown in FIGS. 7 (a) and 7 (b). In FIG. 7 (a), an arm 60 is attached horizontally to a shaft 50 provided in the vertical direction, and a support base 70 for supporting the plate 12 is attached to the tip of the arm 60 so as to be rotatable. It is a thing. In this case, when the shaft 50 rotates, the support base 70 receives the centrifugal force and gradually inclines, and when the rotation speed increases, the support base 70 approaches a state in which it is inclined by 90 ° along the vertical plane from the original state as shown by the alternate long and short dash line. Become. According to this example, the positional relationship between the liquid 13 and the plasma 14 shown in FIG. 3 is always maintained even if the shaft 50 is not rapidly rotated from the beginning. FIG. 7 (b) is the same figure (a).
Arm with the support 70 shown in Fig.
It is attached to the tip of 60. Also in this case, the same effect as the effect of FIG. 7 (a) can be obtained.

EFFECTS OF THE INVENTION According to the present invention, cells can be identified accurately without using complement and while suppressing non-specific reactions.
In this case, it is not necessary to strictly perform pretreatment of the sample blood such as lymphocyte isolation, deproteinization, and serum separation.

According to the present invention, since there is almost no damage to lymphocytes, the present invention can be applied to specimen pretreatment in hematology and immunology studies.

In the present invention, by adjusting the composition and specific gravity of the second liquid, the present invention can be applied to separation, concentration and removal of blood components.

According to the present invention, since automation can be performed by a simple device, cost, time and labor can be reduced.

[Brief description of drawings]

1 is an overall configuration diagram of an apparatus used in the method of the present invention, FIG. 2 is an enlarged view of the plate shown in FIG. 1, and FIG.
And FIG. 2 are explanatory views of the liquid contained in the wells of the plate, FIG. 4 is an explanatory view of the phenomenon inside the well shown in FIG. 3, and FIG. 5 is a schematic view of the well shown in FIG. FIG. 6 is a view showing another example of the shape, FIG. 6 is a view showing various structures in place of the drum shown in FIG. 1, and FIG. 7 is another view showing a mechanism for holding and rotating the plate 12 shown in FIG. It is a figure which shows the example of. 1 ... Drum, 2 ... Support member 3 ... Shaft, 4 ... Arm 5 ... Arm support stand, 6 ... Base stand 7,8 ... Gear, 9 ... Controller 11 ... Well, 12 ... Plate 13 ... Liquid (second liquid) 14 ... Plasma fluid (first liquid) 30 ... Protein A

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) Reference JP-A-63-252252 (JP, A) Special table Sho-63-500962 (JP, A) Special table Sho-62-501233 (JP, A)

Claims (2)

[Claims] 1. A container containing a cell having an antigen-antibody complex formed by binding an antigen and an antibody on the surface of which a substance that specifically binds to an immunoglobulin is fixed to a part of the inner wall of the container. A second liquid having a higher specific gravity than that of the first liquid and containing the substance in the second liquid, and then containing the first liquid on the second liquid; A complex antigen-antibody reaction system in which the container is rotationally moved in a state in which the part of the inner wall is farthest from the center of rotation, the cells are bound to the substance to be solid-phased, and then washed to be identified. Cell identification method in. 2. A first container comprising a container, a substance fixed to a part of an inner wall of the container and specifically binding to an immunoglobulin, and a cell having an antigen-antibody complex formed by binding an antigen and an antibody on the surface thereof. The second liquid having a higher specific gravity than the liquid and stored in the container so as to immerse the substance, and the container is rotated in a state where the part of the inner wall is farthest from the rotation center. A cell immobilization device used for a cell identification method in a complex antigen-antibody reaction system, comprising a container rotating means for moving.